Bisphosphonates inhibit bone resorption and are effective treatments for metabolic bone diseases including osteoporosis and Paget's disease (I. Elomaa, C. Blomqvist, L. Porkka, T. Holmstrom, T. Taube, C. Lamberg-Allardt, G. H. Borgstrom. Lancet 1985; i:1155; J. P. Kosonen, J. Pharm Biomed Anal. 1992; 10:881; J. A. Cantril, H. M. Buckler, D. C. Anderson, Ann. Rheumatol. Dis. 1986; 45:1012; H. Fleisch, Horm. Metab. Res. 1997; 29:145). The prevention of bone resorption results from inhibitory effects on the function of mature osteoclasts. Several bioanalytical methods have been published for bisphosphonates. In general, those methods are mainly based on ion-exchange and ion-pair chromatography with UV, fluorescence (with a pre or post column derivatization), conductivity, flame photometric phosphorus selective, refractive index and explorative light-scattering detection (V. Virtanen, L. H. J. Lajunen, J. Chromatogr. 1993; 617:291; V. Virtanem, L. H. J. Lajunen, Talanta 1993; 40: 661; S. E. Meek, D. J. Pietrzyk Anal. Chem. 1988; 60:1397; M. J. Lovdahl, D. J. Pietrzyk, J. Chromatogr. A 1999; 850:143; R. Niemi, H. Taipale, M. Ahlmark, J. Vepsalainen, T. Jarvinen, J. Chromatogr. B 1997; 701:97; T. L. Chester, Anal. Chem. 1980; 52:1621).
The technique of GC-MS, combined with acylation and silylation, has been used to determine Risedronate in human urine (D. Y. Mitchell, R. A. Eusebio, L. E. Dunlap, K. A. Pallone, J. D. Nesbitt, D. A. Russell, M. E. Clay, P. J. Bekker, Pharm. Res. 1998; 15:228). The more sensitive method for analysis of Risedronate in human urine has been achieved using enzyme linked immunosorbent assay (ELISA) (D. Y. Mitchell, M. A. Heise, K. A. Pallone, J. D. Nesbilt, M. E. Clay, J. D. Nesbitt, D. A. Russell, C. W. Melson, J. Clin. Pharmacol. 1999; 48:536). The column-switching ion-pair HPLC with UV detection has also been reported to quantify the Risedronate in human urine (P. T. Vallano, S. B. Shugars, W. F. Kline, E. J. Woolf, B. K. Matuszewski, J. Chromatogr. B 1003; 794:23). Although some of these methods showed a very high sensitivity, they are all very complicated and time-consuming.
Recently, more effort has been put on the development of LC/MS/MS method for risedronate with post-extraction or on cartridge derivertization (L. S. Zhu, V. N. Lapko, J. W. Lee, Y. J. Basir, C. Kafonek, R. Olsen, C. Briscoe, Rapid commun. Mass Spectrom. 2006, 20: 3421; S. Turcotte, J. Couture, F. Vallee, AAPS PharmSci Vol. 5, No. 4, Abstract M1357 (2003).
The present invention is directed to a method for quantitatively determining risedronate in a urine sample comprising:                a) adding an internal standard to the urine sample;        b) applying the urine sample to an Oasis® HLB cartridge, wherein the cartridge has been pre-conditioned with methanol;        c) washing the cartridge with about 1% (v/v) TEA in water and about 1% (v/v) formic acid in methanol;        d) eluting risedronate, at least once, with a mixture of about 60% (v/v) methanol and about 40% (v/v) water containing about 3 mM EDTA under vacuum;        e) evaporating the eluted solution and reconstituting with a mixture of 10% (v/v) methanol and 90% (v/v) 0.05 M NH4Ac—NH4OH buffer to provide a sample mixture of risedronate and the internal standard; and        f) analyzing the sample mixture with a LC-MS/MS system.        
The aforesaid SPE-LC-MS-MS method for quantitative determination of risedronate in a urine sample is relatively simple, sensitive, precise and accurate, and more fully discussed with the aid of the following figures and detailed description below.